| |
| author |
Gabby Tronosky
| | title |
The Effect of Salt Ions on the Substrate Inhibition of a Novel Aldo-Keto Reductase
| | abstract |
Aldo-Keto Reductases (AKRs) are a superfamily of enzymes that catalyze the reduction of carbonyl compounds to alcohols.
AKRs are expressed in all plants and animals, and appear to play a key role in metabolism and detoxification processes.
These enzymes are highly useful in biocatalysis due to their ability to
facilitate asymmetric reduction, and have been implemented in the synthesis of many drugs,
including the chemotherapy drug Taxol (Feske et al. 2005). Conversely, AKR overexpression has
been linked to various diseases, such as cancer, and thus the inhibition of these enzymes is a
significant topic of research (Qu et al. 2021). Despite their importance in these fields, much
remains unknown about the precise physiological functions of AKRs. This research focuses on
uncovering the workings of a novel Aldo-Keto Reductase, AKR163, discovered in an ancient
yeast strain. Reduction of substrates containing electron withdrawing groups by this enzyme
results in substrate inhibition. The proposed model of inhibition, claims that a second substrate
binds to the enzyme-cofactor complex after reduction, removing it from the reaction equilibrium.
AKRs have been shown throughout literature to bind their cofactors through electrostatic
interactions. In an attempt to determine the validity of the predicted inhibition model, and learn
how to alleviate the substrate inhibition, the ability of various salt ions: NaCl, KCl, Na2SO4, and
NaBr, to decrease the affinity of these interactions, and overall provide less opportunity for
inhibition, was analyzed. To first analyze the effect of a salt on the KM of the cofactor NADPH,
1M NaCl was added to reaction mixtures containing AKR163, Ethyl-4-chloroacetoacetate, and
NADPH in concentrations ranging from 10-200µM. Fitting kinetic data to the Michaelis Menten
equation revealed a 14-fold increase in the Michaelis constant KM, suggesting the decreased
cofactor affinity caused by the ions. To analyze the effect on substrate inhibition, reaction
mixtures containing AKR163, NADPH and Ethyl-4-chloro-acetoacetate (E4ClAA)
concentrations ranging from 30-12000µM were prepared, and their kinetics were analyzed in the
absence of salt, and in the presence of 1M NaCl, 1M KCl, 0.33M Na2SO4 or 1M NaBr. Fitting
data to the substrate inhibition equation reveals an increased inhibition constant Ki in the
presence of each salt compared to a no salt control, correlating with a decreased potency of
inhibition. NaBr appears to provide the most protection, with the largest Ki increase of 10-fold.
This salt, however, resulted in a 20% decrease in the maximum velocity Vmax. Overall the data
implies that salts containing larger ions have a greater protective effect against the substrate
inhibition, but have varying effects on the Vmax and kcat of the reaction. It has overall been found
that the addition of salt to the environment provides protection against substrate inhibition in the
AKR-163 catalyzed reduction of alcohols.
| | school |
The College of Liberal Arts, Drew University
| | degree |
B.S. (2023)
|
| advisor |
Dr. Adam Cassano
|
| full text | GTronosky.pdf |
| |
